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author:("idko, Marco")
1.  Nucleotide signalling during inflammation 
Nature  2014;509(7500):310-317.
Inflammatory conditions are associated with the extracellular release of nucleotides, particularly ATP. In the extracellular compartment, ATP predominantly functions as a signalling molecule through the activation of purinergic P2 receptors. Metabotropic P2Y receptors are G-protein-coupled, whereas ionotropic P2X receptors are ATP-gated ion channels. Here we discuss how signalling events through P2 receptors alter the outcomes of inflammatory or infectious diseases. Recent studies implicate a role for P2X/P2Ysignalling in mounting appropriate inflammatory responses critical for host defence against invading pathogens or tumours. Conversely, P2X/P2Y signalling can promote chronic inflammation during ischaemia and reperfusion injury, inflammatory bowel disease or acute and chronic diseases of the lungs. Although nucleotide signalling has been used clinically in patients before, research indicates an expanding field of opportunities for specifically targeting individual P2 receptors for the treatment of inflammatory or infectious diseases.
PMCID: PMC4222675  PMID: 24828189
2.  CD39 is a negative regulator of P2X7-mediated inflammatory cell death in mast cells 
Mast cells (MCs) are major contributors to an inflammatory milieu. One of the most potent drivers of inflammation is the cytokine IL-1β, which is produced in the cytoplasm in response to danger signals like LPS. Several controlling mechanisms have been reported which limit the release of IL-1β. Central to this regulation is the NLRP3 inflammasome, activation of which requires a second danger signal with the capacity to subvert the homeostasis of lysosomes and mitochondria. High concentrations of extracellular ATP have the capability to perturb the plasma membrane by activation of P2X7 channels and serve as such a danger signal. In this study we investigate the role of P2X7 channels and the ecto-5´-nucleotidase CD39 in ATP-triggered release of IL-1β from LPS-treated mast cells.
We report that in MCs CD39 sets an activation threshold for the P2X7-dependent inflammatory cell death and concomitant IL-1β release. Knock-out of CD39 or stimulation with non-hydrolysable ATP led to a lower activation threshold for P2X7-dependent responses. We found that stimulation of LPS-primed MCs with high doses of ATP readily induced inflammatory cell death. Yet, cell death-dependent release of IL-1β yielded only minute amounts of IL-1β. Intriguingly, stimulation with low ATP concentrations augmented the production of IL-1β in LPS-primed MCs in a P2X7-independent but caspase-1-dependent manner.
Our study demonstrates that the fine-tuned interplay between ATP and different surface molecules recognizing or modifying ATP can control inflammatory and cell death decisions.
PMCID: PMC4110707  PMID: 25184735
ATP; BMMC; CD39; Cell death; IL-1β; P2X7
3.  The Nlrp3 inflammasome regulates acute graft-versus-host disease 
The Journal of Experimental Medicine  2013;210(10):1899-1910.
Conditioning therapies before transplantation induce the release of uric acid, which triggers the NLRP3 inflammasome and IL-1β production contributing to graft-versus-host disease.
The success of allogeneic hematopoietic cell transplantation is limited by acute graft-versus-host disease (GvHD), a severe complication accompanied by high mortality rates. Yet, the molecular mechanisms initiating this disease remain poorly defined. In this study, we show that, after conditioning therapy, intestinal commensal bacteria and the damage-associated molecular pattern uric acid contribute to Nlrp3 inflammasome–mediated IL-1β production and that gastrointestinal decontamination and uric acid depletion reduced GvHD severity. Early blockade of IL-1β or genetic deficiency of the IL-1 receptor in dendritic cells (DCs) and T cells improved survival. The Nlrp3 inflammasome components Nlrp3 and Asc, which are required for pro–IL-1β cleavage, were critical for the full manifestation of GvHD. In transplanted mice, IL-1β originated from multiple intestinal cell compartments and exerted its effects on DCs and T cells, the latter being preferentially skewed toward Th17. Compatible with these mouse data, increased levels of active caspase-1 and IL-1β were found in circulating leukocytes and intestinal GvHD lesions of patients. Thus, the identification of a crucial role for the Nlrp3 inflammasome sheds new light on the pathogenesis of GvHD and opens a potential new avenue for the targeted therapy of this severe complication.
PMCID: PMC3782050  PMID: 23980097
4.  Modified Foxp3 mRNA protects against asthma through an IL-10–dependent mechanism  
The Journal of Clinical Investigation  2013;123(3):1216-1228.
Chemically modified mRNA is capable of inducing therapeutic levels of protein expression while circumventing the threat of genomic integration often associated with viral vectors. We utilized this novel therapeutic tool to express the regulatory T cell transcription factor, FOXP3, in a time- and site-specific fashion in murine lung, in order to prevent allergic asthma in vivo. We show that modified Foxp3 mRNA rebalanced pulmonary T helper cell responses and protected from allergen-induced tissue inflammation, airway hyperresponsiveness, and goblet cell metaplasia in 2 asthma models. This protection was conferred following delivery of modified mRNA either before or after the onset of allergen challenge, demonstrating its potential as both a preventive and a therapeutic agent. Mechanistically, FOXP3 induction controlled Th2 and Th17 inflammation by regulating innate immune cell recruitment through an IL-10–dependent pathway. The protective effects of FOXP3 could be reversed by depletion of IL-10 or administration of recombinant IL-17A or IL-23. Delivery of Foxp3 mRNA to sites of inflammation may offer a novel, safe therapeutic tool for the treatment of allergic asthma and other diseases driven by an imbalance in helper T cell responses.
PMCID: PMC3582134  PMID: 23391720
5.  AMP Affects Intracellular Ca2+ Signaling, Migration, Cytokine Secretion and T Cell Priming Capacity of Dendritic Cells 
PLoS ONE  2012;7(5):e37560.
The nucleotide adenosine-5′-monophosphate (AMP) can be released by various cell types and has been shown to elicit different cellular responses. In the extracellular space AMP is dephosphorylated to the nucleoside adenosine which can then bind to adenosine receptors. However, it has been shown that AMP can also activate A1 and A2a receptors directly. Here we show that AMP is a potent modulator of mouse and human dendritic cell (DC) function. AMP increased intracellular Ca2+ concentration in a time and dose dependent manner. Furthermore, AMP stimulated actin-polymerization in human DCs and induced migration of immature human and bone marrow derived mouse DCs, both via direct activation of A1 receptors. AMP strongly inhibited secretion of TNF-α and IL-12p70, while it enhanced production of IL-10 both via activation of A2a receptors. Consequently, DCs matured in the presence of AMP and co-cultivated with naive CD4+CD45RA+ T cells inhibited IFN-γ production whereas secretion of IL-5 and IL-13 was up-regulated. An enhancement of Th2-driven immune response could also be observed when OVA-pulsed murine DCs were pretreated with AMP prior to co-culture with OVA-transgenic naïve OTII T cells. An effect due to the enzymatic degradation of AMP to adenosine could be ruled out, as AMP still elicited migration and changes in cytokine secretion in bone-marrow derived DCs generated from CD73-deficient animals and in human DCs pretreated with the ecto-nucleotidase inhibitor 5′-(alpha,beta-methylene) diphosphate (APCP). Finally, the influence of contaminating adenosine could be excluded, as AMP admixed with adenosine desaminase (ADA) was still able to influence DC function. In summary our data show that AMP when present during maturation is a potent regulator of dendritic cell function and point out the role for AMP in the pathogenesis of inflammatory disorders.
PMCID: PMC3356328  PMID: 22624049
6.  Lack of the purinergic receptor P2X7 results in resistance to contact hypersensitivity 
The Journal of Experimental Medicine  2010;207(12):2609-2619.
Engagement of P2X7 on mouse dendritic cells, presumably by ATP released in response to contact allergen, is needed for IL-1β production and the sensitization phase of contact hypersensitivity.
Sensitization to contact allergens requires activation of the innate immune system by endogenous danger signals. However, the mechanisms through which contact allergens activate innate signaling pathways are incompletely understood. In this study, we demonstrate that mice lacking the adenosine triphosphate (ATP) receptor P2X7 are resistant to contact hypersensitivity (CHS). P2X7-deficient dendritic cells fail to induce sensitization to contact allergens and do not release IL-1β in response to lipopolysaccharide (LPS) and ATP. These defects are restored by pretreatment with LPS and alum in an NLRP3- and ASC-dependent manner. Whereas pretreatment of wild-type mice with P2X7 antagonists, the ATP-degrading enzyme apyrase or IL-1 receptor antagonist, prevents CHS, IL-1β injection restores CHS in P2X7-deficient mice. Thus, P2X7 is a crucial receptor for extracellular ATP released in skin in response to contact allergens. The lack of P2X7 triggering prevents IL-1β release, which is an essential step in the sensitization process. Interference with P2X7 signaling may be a promising strategy for the prevention of allergic contact dermatitis.
PMCID: PMC2989767  PMID: 21059855
7.  5-Hydroxytryptamine Modulates Migration, Cytokine and Chemokine Release and T-Cell Priming Capacity of Dendritic Cells In Vitro and In Vivo 
PLoS ONE  2009;4(7):e6453.
Beside its well described role in the central and peripheral nervous system 5-hydroxytryptamine (5-HT), commonly known as serotonin, is also a potent immuno-modulator. Serotoninergic receptors (5-HTR) are expressed by a broad range of inflammatory cell types, including dendritic cells (DCs). In this study, we aimed to further characterize the immuno-biological properties of serotoninergic receptors on human monocyte-derived DCs. 5-HT was able to induce oriented migration in immature but not in LPS-matured DCs via activation of 5-HTR1 and 5-HTR2 receptor subtypes. Accordingly, 5-HT also increased migration of pulmonary DCs to draining lymph nodes in vivo. By binding to 5-HTR3, 5-HTR4 and 5-HTR7 receptors, 5-HT up-regulated production of the pro-inflammatory cytokine IL-6. Additionally, 5-HT influenced chemokine release by human monocyte-derived DCs: production of the potent Th1 chemoattractant IP-10/CXCL10 was inhibited in mature DCs, whereas CCL22/MDC secretion was up-regulated in both immature and mature DCs. Furthermore, DCs matured in the presence of 5-HT switched to a high IL-10 and low IL-12p70 secreting phenotype. Consistently, 5-HT favoured the outcome of a Th2 immune response both in vitro and in vivo. In summary, our study shows that 5-HT is a potent regulator of human dendritic cell function, and that targeting serotoninergic receptors might be a promising approach for the treatment of inflammatory disorders.
PMCID: PMC2714071  PMID: 19649285
8.  Inhaled iloprost suppresses the cardinal features of asthma via inhibition of airway dendritic cell function 
Journal of Clinical Investigation  2007;117(2):464-472.
Inhalation of iloprost, a stable prostacyclin (PGI2) analog, is a well-accepted and safe treatment for pulmonary arterial hypertension. Although iloprost mainly acts as a vasodilator by binding to the I prostanoid (IP) receptor, recent evidence suggests that signaling via this receptor also has antiinflammatory effects through unclear mechanisms. Here we show in a murine model of asthma that iloprost inhalation suppressed the cardinal features of asthma when given during the priming or challenge phase. As a mechanism of action, iloprost interfered with the function of lung myeloid DCs, critical antigen-presenting cells of the airways. Iloprost treatment inhibited the maturation and migration of lung DCs to the mediastinal LNs, thereby abolishing the induction of an allergen-specific Th2 response in these nodes. The effect of iloprost was DC autonomous, as iloprost-treated DCs no longer induced Th2 differentiation from naive T cells or boosted effector cytokine production in primed Th2 cells. These data should pave the way for a clinical effectiveness study using inhaled iloprost for the treatment of asthma.
PMCID: PMC1783814  PMID: 17273558
9.  Local application of FTY720 to the lung abrogates experimental asthma by altering dendritic cell function 
Journal of Clinical Investigation  2006;116(11):2935-2944.
Airway DCs play a crucial role in the pathogenesis of allergic asthma, and interfering with their function could constitute a novel form of therapy. The sphingosine 1–phosphate receptor agonist FTY720 is an oral immunosuppressant that retains lymphocytes in lymph nodes and spleen, thus preventing lymphocyte migration to inflammatory sites. The accompanying lymphopenia could be a serious side effect that would preclude the use of FTY720 as an antiasthmatic drug. Here we show in a murine asthma model that local application of FTY720 via inhalation prior to or during ongoing allergen challenge suppresses Th2-dependent eosinophilic airway inflammation and bronchial hyperresponsiveness without causing lymphopenia and T cell retention in the lymph nodes. Effectiveness of local treatment was achieved by inhibition of the migration of lung DCs to the mediastinal lymph nodes, which in turn inhibited the formation of allergen-specific Th2 cells in lymph nodes. Also, FTY720-treated DCs were intrinsically less potent in activating naive and effector Th2 cells due to a reduced capacity to form stable interactions with T cells and thus to form an immunological synapse. These data support the concept that targeting the function of airway DCs with locally acting drugs is a powerful new strategy in the treatment of asthma.
PMCID: PMC1626118  PMID: 17080194
10.  P2X7 receptor: Death or life? 
Purinergic Signalling  2005;1(3):219-227.
The P2X7 plasma membrane receptor is an intriguing molecule that is endowed with the ability to kill cells, as well as to activate many responses and even stimulate proliferation. Here, the authors give an overview on the multiplicity and complexity of P2X7-mediated responses, discussing recent information on this receptor. Particular attention has been paid to early and late signs of apoptosis and necrosis linked to activation of the receptor and to the emerging field of P2X7 function in carcinogenesis.
PMCID: PMC2096546  PMID: 18404507
cell death; inflammation; P2X7; proliferation
11.  Stimulation of P2 purinergic receptors induces the release of eosinophil cationic protein and interleukin-8 from human eosinophils 
British Journal of Pharmacology  2003;138(7):1244-1250.
Extracellular nucleotides are the focus of increasing attention for their role as extracellular mediators since they are released into the extracellular environment in a regulated manner and/or as a consequence of cell damage.Here, we show that human eosinophils stimulated with different nucleotides release eosinophil cationic protein (ECP) and the chemokine interleukin 8 (IL-8), and that release of these two proteins has a different nucleotide requirement.Release of ECP was triggered in a dose-dependent manner by ATP, UTP and UDP, but not by 2′-&3′-o-(4-benzoyl-benzoyl)adenosine 5′-triphosphate (BzATP), ADP and α,β-methylene adenosine 5′ triphosphate (α,β-meATP). Release of IL-8 was triggered by UDP, ATP, α,β-meATP and BzATP, but not by UTP or ADP. Pretreatment with pertussis toxin abrogated nucleotide-stimulated ECP but not IL-8 release.Release of IL-8 stimulated by BzATP was fully blocked by the P2X7 blocker KN-62, while release triggered by ATP was only partially inhibited. IL-8 secretion due to UDP was fully insensitive to KN-62 inhibition.Priming of eosinophils with GM-CSF increased IL-8 secretion irrespectively of the nucleotide used as a stimulant.It is concluded that extracellular nucleotides trigger secretion of ECP by stimulating a receptor of the P2Y subfamily (possibly P2Y2), while, on the contrary, nucleotide-stimulated secretion of IL-8 can be due to activation of both P2Y (P2Y6) and P2X (P2X1 and P2X7) receptors.
PMCID: PMC1573766  PMID: 12711624
Human eosinophils; extracellular ATP; P2 receptors; ECP; IL-8

Results 1-11 (11)